Project Summary Evasion of apoptosis is a hallmark of cancer and is associated with oncogenesis, tumor maintenance, and resistance to conventional chemotherapy. BCL-2 was the first identified anti-apoptotic factor and is a member of the BCL-2 family of proteins that cooperate in the regulation of programmed cell death. BCL-2 plays a dominant role in the survival of multiple lymphoid malignancies and has consequently become an active target for drug discovery programs seeking to reactivate dormant apoptotic signaling in cancer cells or enhance the activity of cytotoxic anticancer drugs. The only small-molecule-based approach has been to target the BH3 binding domain of Bcl-2, thereby blocking interaction with other pro-apoptotic Bcl-2 family members. However, the recent suspension of the BH3 mimetic ABT-119 from clinical trials, due to the occurrence of tumor lysis syndrome, has cast some doubt over the potential safety of the BH3 mimetic approach and increased the need for alternative strategies to target BCL-2. In common with many other genes that cooperate in the development and maintenance of cancer, BCL-2 contains a highly plastic, GC-rich transcriptional regulatory element in its promoter region. Pioneering work at the University of Arizona has shown that this regulatory element is capable of forming DNA secondary structures called G-quadruplex DNA (G-rich strand) and i-motif DNA (C-rich strand) and that these DNA secondary structures regulate transcription. A high-throughput screening assay designed to identify small molecules capable of stabilizing/destabilizing the BCL-2 i-motif sequence produced a small molecule from the steroidal chemotype (IMC-76). Subsequent studies have demonstrated that IMC-76 decreases BCL-2 expression in cell culture and in mouse xenograft tumor cells. Importantly, IMC-76 significantly potentiates apoptosis induced by conventional chemotherapy drugs in cell culture and demonstrates considerable synergy when combined with chemotherapy drugs in mouse cancer xenograft models. The ultimate goal of this proposal is to advance suitably optimized analogs of IMC-76 into IND-enabling studies and eventual phase 1 clinical trials. We propose a limited round of synthetic chemistry for the purpose of optimizing potency for lowering BCL-2 expression in cells and synergy with cytotoxic anticancer drugs in cell culture. Furthermore, this optimization strategy will allow us to explore potential off-target effects associated with the steroidal chemotype by screening analogs in biochemical nuclear receptor assays. Qualified lead molecules will be subjected to pharmacokinetic analysis in rats, both chronic and acute tolerability studies in mice, and efficacy studies in combination with standard-of-care chemotherapy drugs in lymphoma mouse xenograft models. Collectively these studies will enable us to select an optimized lead candidate for advancement into formal preclinical studies.